Flavobacterium psychrophilum est le pathogène de la maladie de l’eau froide ou du syndromede l’alevin qui provoque des pertes économiques importantes dans les élevages de truite arc-en-cielen Aquitaine. Au cours de cette thèse, nous avons développé dans un premier temps un nouveau testqPCR TaqMan en duplex qui cible les gènes (i) adnr16S de F. psychrophilum et (ii) β-actine de latruite arc-en-ciel ; il permet de déterminer le taux d’infection de tissus contaminés au cours d’unemême analyse.Dans un second temps, nous avons cherché à objectiver trois approches préventives quipourraient être mises en oeuvre pour limiter l’infection à F. psychrophilum dans les élevages. Lacaractérisation de biocides classiquement utilisés par la filière aquacole a permis de proposer auxaquaculteurs des méthodes de désinfection réellement efficaces contre F. psychrophilum mais quin’affectent pas le développement des oeufs ni des futurs alevins. En parallèle, l’impact de la mise enplace de traitement probiotiques a été caractérisé à la surface des oeufs, à la surface du mucus et/oule long du système digestif, dès les tout premiers stades de développement des truites, sur le nombreet l’intensité des crises de flavobactériose dans les élevages. Deux traitements microbiens ont étéretenus : l’E112® (DOXAL-FRANCE, Pessac) et le Bactocell® (LALLEMAND, Toulouse). Enfin, troispréparations vaccinales ont été préparées (« Adjuvant », « Autovaccin » et « Membrane Externe ») ettestées pour leur capacité à protéger les poissons de la flavobactériose. / Flavobacterium psychrophilum is the causative agent of cold water disease or the rainbow troutfry syndrome which causes important economic losses in rainbow trout breedings. During this thesis,we firstly developed a new duplex Taqman qPCR test which targets the following genes (i) F.psychrophilum adnr16S and (ii) rainbow trout β-actine. This test enables to obtain the infection rate ofcontaminated tissues, during a same analysis.Then, we tried to objectivize three preventive approaches which could be implemented inbreedings to limit the infection by F. psychrophilum. The characterization of biocides used inaquaculture allowed proposing to the fish farmers effective methods of disinfection against F.psychrophilum. These methods do not affect the development of eggs or future fish larva. Moreover,the impact of the implementation of probiotics on the surface of eggs, on the surface of the mucusand/or the digestive system, from the very first steps of trout development has been characterized onthe number and the intensity of flavobacteriosis outbreaks in the breeding farms. Two probiotictreatments were selected: l’E112® (DOXAL-FRANCE, Pessac) and Bactocell® (LALLEMAND, Toulouse).Finally, three vaccine preparations (« Adjuvant », « Auto-vaccine » and « Outer Membrane ») wereprepared and tested for their capability to protect farmed fish against flavobacteriosis.

This study investigated the regulation of Na⁺ Ca²⁺ Cl⁻ during development in embryonic rainbow trout (Oncorhynchus mykiss). Because there is a close relationship between pH regulation and ion uptake mechanisms in adult teleosts, pH, pCO₂, and NH₄⁺ levels in unstirred layers (USLs) adjacent to whole eggs and dechorionated embryos were determined using double-barrel ion-selective microelectrodes (ISMEs). Whole eggs accumulated Na⁺, Ca²⁺ and Cl⁻ during the last 20 days of embryonic development, suggesting an ionoregulatory ability prior to hatching. Na⁺ uptake by whole eggs was linearly related to external [Na⁺], suggesting that Na⁺ crosses the chorion by diffusion. The uptake by dechorionated embryos was saturable, indicating the presence of active transport or facilitated diffusion mechanisms on the surface of embryos prior to hatching. Ca²⁺ uptake by whole eggs and dechorionated embryos was saturable, suggesting that specific pathways or binding sites are present in the chorion, and that active transport or facilitated diffusion mechanisms are present at the surface of embryos. The much higher Jₘₐₓ for whole eggs than dechorionated embryos suggests a role of the perivitelline fluid (pvf) or chorion in ion uptake. Jₘₐₓ was lower in dechorionated embryos than in hatchlings suggesting that Ca²⁺ uptake mechanisms may not be fully developed in dechorionated embryos. Low pH and high [NH₄⁺] were measured in the USLs adjacent to whole eggs and dechorionated embryos, relative to the bulk water. A high Pcₒ₂ measured adjacent to the gills suggests that CO₂ excretion was the primary source of USL acidification. pH was lower in the USL adjacent to the gills and trunk than in the USL adjacent to the yolk sac. [NH₄⁺] was highest adjacent to the gills and trunk than adjacent to the yolk sac. There was no change in the extent of USL acidification or in [NH₄⁺] over the last half of embryonic development. After hatching, however, there was a significant increase in [NH₄⁺] adjacent to the gills, which was not accompanied by a change in USL pH. It is possible that a change in permeability of the transporting surfaces (i.e. gills, trunk, yolk sac) occurs after hatching. / Thesis / Master of Science (MS)

Acute ammonia toxicity in rainbow trout was studied. This was carried out by injecting fish with various concentrations of ammonia dissolved in isotonic saline. In order to approximate conditions of natural toxicity, where ammonia would enter the fish at the gills, without the additional problems of environmental factors, the ammonium solutions were injected via a cannula implanted in the dorsal aorta. To determine if a differential toxicity existed in fish in relation to high pH and low pH ammonium solutions, ammonium bicarbonate and ammonium chloride were chosen for injection. Hydrogen ion, and total ammonia, concentrations were measured in blood sampled from the dorsal aorta, both before and after injection. In order to determine if, during the course of the injection, normal excretion rates would remove all of the injected ammonia, total ammonia and hydrogen ion concentrations were measured in blood sampled from both the dorsal and ventral aortae, and rates of extraction of ammonia from blood at the gills were calculated. Ammonia is toxic to fish. There was no significant difference between the dose of NH₄Cl and NH₄HCO₃ which killed fish. Therefore, unlike mammals, fish exhibited no differential toxicity to the ammonium compounds tested. Injection of NH₄Cl decreased pHa and injection of NH₄HCO₃ increased pHa. Both compounds increased the total ammonia concentration in the blood. Although in water the fraction of ammonia which exerts the toxic effects is unionized ammonia, within the fish it is the ionized fraction which exerts the toxic effects. The same dose of ammonium killed fish, but NH₄HCO₃-injected fish which survived had a much higher concentration of unionized ammonia in the blood than NH₄Cl-injected fish which died. Ammonia extracted from the blood in control fish was about one-fifth the amount which killed fish. This, together with the measured increases in blood ammonia following injection, demonstrate that, although ammonia is a normal excretory product of rainbow trout, the trout cannot increase excretion rates sufficiently to rid themselves of an ammonia load. Symptoms observed in fish following injection of ammonium solutions led to the conclusion that ammonia acts at the neural level. / Science, Faculty of / Zoology, Department of / Graduate

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 275-310). Also available on the World Wide Web.

Co-evolution between parasites and their hosts result in extremely well-orchestrated and intimate relationships that are characterized by remarkable adaptations in the attack response of the parasite and the defense response of the host. To fully understand host-parasite interactions, these adaptations must be considered in the context of the ecological constraints in which they evolved. As a serious pest to salmon mariculture, Lepeophtheirus salmonis has been extensively studied; however, there are still several areas that require further research. Of utmost importance, and the topic of this thesis, is molecular basis for resistance to sea lice. The following chapters investigate this phenomena under the umbrella of ecological immunology using combined modern technologies of transcriptomics, proteomics and functional immunology with a focus on the primary interaction site. In the first chapter, I describe the key players involved in this host-parasite relationship with a focus on the primary interaction site, the louse-salmon interface, where there are responses by the louse (attack) and the salmon host (defense). Previous research indicated that an early aggressive inflammatory response at the louse-skin interface contributes to resistance in coho salmon; however, there are no data characterizing a site-specific response in resistant (pink and coho) and susceptible (Atlantic, chum) species. Accordingly in Chapter 2, I define site-specific cutaneous responses in Atlantic, pink and chum salmon to establish genetic biomarkers of resistance. Chapter 3 focuses on identification of cellular effectors using histochemical localization of biomarkers to characterize cellular populations activated at the louse-attachment site, while broadening the gene targets. Our notion of pink salmon as a resistant species is challenged by the common observation of migrating pink salmon supporting large populations of L. salmonis in the field. Thus the purpose of chapter 4 was to investigate potential mechanisms to explain variations in susceptibility as a function of life history. Host-parasite relationships are a product of both host and parasite responses; therefore, in chapters 5 and 6, I shift focus to the level of the parasite. In chapter 5 I present the first documented large-scale transcriptomic profiling of L. salmonis during feeding on both resistant (coho) and susceptible (Atlantic, sockeye) salmon. This was followed (chapter 6) by describing the proteomic profile of L. salmonis secretions after feeding on Atlantic salmon. In the seventh and final chapter, I present my conclusions on the molecular mechanisms for resistance to sea lice and discuss potential applications of this information for future louse control strategies. / Graduate

Mitochondria were isolated from heart and mosaic muscle of rainbow trout (Salmo qairdneri R.). State 3 respiratory rates were determined at 5 and 15°C using pyruvate, malate, lactate, glutamate or acetyl—carnitine as substrate. The final three substrates were used to generate pH profiles. Pyruvate was oxidized at high rates in all cases, indicating good potential for aerobic carbohydrate metabolism. At 15°C, malate was an equally good substrate for heart mitochondria, while all substrates were oxidized at similar rates to pyruvate in muscle mitochondria. Maximal oxidation rates of heart mitochondria were greater than or equal to those of muscle. State 3 Q₁₀ for oxidation of most substrates in heart was approximately 2, except for malate which had a Q₁₀ of 3. Mitochondrial oxidation tended to be more sensitive to decreased temperature in muscle than in heart, particularly with respect to acetyl—carnitine and glutamate oxidation which in muscle had Q₁₀ values of 4 and 7, respectively. Based on RCR values at 5 and 15°C, there was no indication that membrane permeability to H⁺ ions was altered by a 10°C change in temperature in mitochondria from either tissue. At pH above 7.6 respiratory rates decreased with increasing pH. State 3 respiratory rate increased in heart mitochondria as pH decreased, below 7.6 while in muscle mitochondria, no such pH dependence was observed. RCR values were above 4 in all experiments except at high pH. Muscle mitochondria were the more sensitive to extreme pH with respect to RCR. Heart mitochondria had higher oxidative rates than those of muscle and were less sensitive to decreased temperature, in keeping with the greater oxidative demands of that tissue relative to mosaic muscle. Muscle mitochondria which typically face larger fluctuations in extramitochondrial pH in vivo than do those of heart, were less sensitive to pH in vitro. It was concluded that substrate utilization patterns and response to changes in temperature and extramitochondrial pH in the two mitochondrial populations was different and reflected both the intracellular environment of the mitochondria and the different needs of each tissue for aerobic energy supply. / Science, Faculty of / Zoology, Department of / Graduate

The role of carbonic anhydrase in carbon dioxide excretion and acid-base regulation in the rainbow trout, Salmo gairdneri has been investigated. While a significant amount of carbonic anhydrase was found in the blood of the trout, calculations based on red cell hemolysates suggest that the probable circulating levels of carbonic anhydrase activity in blood may not be sufficient to account for the observed carbon dioxide excretion. An analysis of carbonic anhydrase activity in whole blood from the trout revealed that intact fish erythrocytes, unlike mammalian erythrocytes totally fail to facilitate the dehydration of extracellular bicarbonate. The possible mechanism of this phenomenon has been examined; however the salient point was that fish red blood cells do not appear capable and therefore by implication apparently not necessary for the excretion of carbon dioxide at the gills of trout. The observed excretion of carbon dioxide in the trout was found to be accounted for by the gills and their compliment of carbonic anhydrase. This finding was based on the following observations. (1) Depletion of circulating blood carbonic anhydrase levels during severe anemia was without effect on carbon dioxide excretion rates or blood acid-base status. (2) Introduction of the carbonic anhydrase inhibitor, diamox into anemic fish produced a severe acid-base disturbance associated with a fall in observed carbon dioxide excretion. (3) Isolated perfused gill preparations excrete carbon dioxide at rates comparable to those observed in vivo from free swimming fish. (4) Carbon dioxide excretion in isolated gill preparations is abolished by diamox. The excretion of carbon dioxide in fish occurs via the movement of plasma bicarbonate into the branchial epithelium, where it is subsequently dehydrated into molecular carbon dioxide and excreted. A model is proposed and supportive evidence presented to account for the coupling of ionic exchange occurring across the gill with carbon dioxide excretion. The proposed model distinguishes between control of plasma hydrogen ion activity and regulation of plasma total carbon dioxide concentration per se. The functional significance of this pattern of carbon dioxide excretion for aquatic animals is discussed along with the implications for air breathing fish. / Science, Faculty of / Zoology, Department of / Graduate

<p> Knowledge into uptake rate and interactions of the metals via the gastrointestinal tract of freshwater fish is vital, in order to provide tools to protect and to sustain aquatic biota. An in vitro stomach and gut sac technique was utilized to investigate uptake rates of essential metals (copper, zinc and nickel) and non-essential metals (cadmium, lead and nickel) at luminal concentrations of 50fJ.M via the gastrointestinal tract Metals had no effect on the fluid transport rates via the gastrointestinal tract except for copper at the stomach. The stomach emerged as small but important site for metal absorption and interaction. Essential metals were absorbed at approximately the same rate as nonessential metals via the gastrointestinal tract. Copper, zinc, nickel, silver, and lead showed statistical correlation between rate of absorption and mucus binding via gastrointestinal tract, an important first finding for the development of a Biotic Ligand Model (BLM) for the gastrointestinal tract of trout. </p> <p> There was an antagonistic effect of calcium on cadmium uptake at the stomach but not at the intestine. Zinc and calcium exhibited synergistic interaction at the stomach but no interaction at the intestine. These results showed the possibility of the transporters DMTl to mediate copper and cadmium uptake via the intestine; hZip 2 to mediate copper and zinc uptake at the stomach; and Mzip 4 or ZTLl to mediate zinc uptake via the gastrointestinal tract of trout. These results can be used to develop a BLM for the gastrointestinal tract of fish. </p> / Thesis / Master of Science (MSc)

<p> Juvenile rainbow trout (Oncorhynchus myldss) were chronically exposed (90 days) in synthetically softened water ([Ca2+]=50, [Na+]=100 J.tequiv·L-1), to sublethal low pH (5.2) and a simulated global warming scenario (+20C), added to the natural summer thermal cycle ofinshore Lake Ontario. Two studies were conducted over the periods June September 1993 and 1994 in order to examine the effects ofthe sublethal stressors under conditions ofunlimited food ration (satiation feeding twice daily= 10% dry body weight·day-1), and a limited food ration (4% dry body weight·day-1) respectively. The addition of 2°C and sulphuric acid was designed to result in four treatment conditions: i) Control conditions; ambient water temperature and pH; ii) ambient water temperature and pH 5.2; iii) simulated global warming at ambient pH; iv) a combination of simulated global warming and pH 5.2. Year to year variation in temperature provided the trout in the satiation-fed study with an ambient ~peraturerange of 13-24°C, while those in the limited ration study experienced a range of 16.5-21°C. Consequently, the trout in the treatments with an additional 2°C experienced temperatures close to the upper incipient lethal level, particularly the trout in the satiation feeding study. Apparent specific dynamic action raised routine metabolic rates in all treatments to -75% and 55% M02(max) in the satiation and limited ration studies respectively, the difference of 20% indicating the influence of an unlimited feeding regime on metabolism. Trout in the satiation feeding study increased in wet body mass by 30-50 g, while trout in the limited ration study increased by only 3-4 g. Whole body proximate composition of the trout fed to satiation changed over time with large increases in lipid content, small increases in protein content, and compensating decreases in water content in all treatments. No such changes occurred in the limited ration trout, although whole body lipid and carbohydrate were highly variable. In both studies, the addition of20C resulted in decreased growth, with an accompanying depression in appetite in the satiation fed trout, especially at peak temperatures. Surprisingly, trout exposed to low pH alone exhibited improved growth in both treatments. Energy budgets indicated that the addition of 2°C reduced gross energy intake and increased fecal (and Ulllidentified) energy losses resulting in lowered conversion efficiencies, while in limited ration trout, energy expenditure was slightly higher. Trout exposed to low pH exhibited higher gross energy intake and gain, and more efficient energy conversion under unlimited food conditions while trout with limited rations expended the least metabolic energy and exhibited lower nitrogen energy losses. The surprising lack of ionoregulatory disturbance in these pH 5.2 exposed trout in both studies suggests tbat the availability of NaCl in the diet was compensating for branchial ion losses, and perhaps driving appetite in the satiation fed trout Where ration was limited, reductions in activity level may have contributed to energy conservation and consequently improved growth. A 22Na+ flux experiment conducted at the end of the limited ration exposure, in which the fish were exposed to a challenge concentration of H+ (pH 4.2), provided evidence for improved recovery of ionoregulatory balance in trout which had been chronically exposed to low pH. Overall, the combination ofincreased global temperatures and sublethal low pH results in increased physiological costs for juvenile rainbow trout, most noticeably when summer temperatures peak. Ration level is of integral importance when considering the degree of impact of such environmental conditions. </p> / Thesis / Master of Science (MSc)

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 118-131).